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N D Greene

Publications and source records attributed to N D Greene.

At least 19 recordsLinked to original sources
PubMed2001

Cardiovascular defects associated with abnormalities in midline development in the Loop-tail mouse mutant.

Loop-tail (Lp) is a naturally occurring mouse mutant that develops severe neural tube defects. In this study, we describe complex cardiovascular defects in Lp homozygotes, which include double-outlet right ventricle, with obligatory perimembranous ventricular septal defects, and double-sided aortic arch, with associated abnormalities in the aortic arch arteries. Outflow tract and aortic arch defects are often related to abnormalities in the cardiac neural crest, but using molecular and anatomic markers, we show that neural crest migration is normal in Lp/Lp embryos. On the other hand, the heart fails to loop normally in Lp/Lp embryos, in association with incomplete axial rotation and reduced cervical flexion. As a consequence, the ventricular loop is shifted posteromedially relative to its position in wild-type embryos. This suggests that the observed cardiac alignment defects in the Lp mutant may be secondary to failure of neural tube closure and incomplete axial rotation. Double-sided aortic arch is a rare finding among mouse models. In humans, it is usually an isolated malformation, only rarely occurring in combination with other cardiac defects. We suggest that the double-sided arch arises as a primary defect in the Lp mutant, unrelated to the alignment defects, perhaps reflecting a role for the (as-yet-unknown) Lp gene in maintenance/regression of the aortic arch system.

Animals↗
PubMed2001

Analysis of CLN3-protein interactions using the yeast two-hybrid system.

Juvenile neuronal ceroid lipofuscinosis (Batten disease) is a childhood neurodegenerative disease that is caused by mutations in the CLN3 gene. The protein encoded by CLN3 has no homology with any proteins of known function and its cellular role remains elusive. In order to investigate the role played by the CLN3 protein we aimed to identify interacting proteins. Here, we describe the yeast two-hybrid system as the approach taken to investigate such protein-protein interactions. CLN3 was expressed as a fusion protein with a DNA-binding domain and used to screen a library of human fetal brain cDNAs fused to a transcriptional activation domain. Owing to low level expression of the full length CLN3 fusion protein, truncated regions corresponding to the predicted hydrophilic regions were also tested. No proteins that interact with CLN3 were detected, nor was there any evidence for CLN3-CLN3 interactions. Potential interaction of CLN3 with subunit c of mitochondrial ATP synthase, the major component of the storage material that accumulates in Batten disease patients, was also tested. No interaction was detected suggesting that the accumulation of subunit c does not result from loss of a process that requires a direct interaction with CLN3. We conclude that either CLN3 does not interact with other proteins or such interactions cannot be detected using the two-hybrid system.

Child↗
PubMed2001

High resolution MRI reveals global changes in brains of Cln3 mutant mice.

Batten disease, the juvenile-onset form of neuronal ceroid lipofuscinosis (NCL), is a progressive neurodegenerative disorder of childhood with an age of onset of 5-10 years of age. JNCL is caused by mutations in the CLN3 gene which encodes a membrane protein of unknown function. Magnetic resonance imaging of the brain of juvenile NCL patients has revealed changes in signal intensity and tissue atrophy, predominantly in the cortex and cerebellum. A mouse model for Batten disease was created by targeted disruption of the murine Cln3 gene in order to further understanding of the pathophysiology of Batten disease and to evaluate potential therapeutic approaches. Several features of the disease are displayed by Cln3 mice including accumulation of characteristic storage material in neurons. The aim of this work was to investigate neurodegeneration in the Cln3 mouse model using high resolution magnetic resonance imaging to measure signal intensity ratios in selected regions of interest. Global changes were observed in the brains of 12-month-old mutant mice that mirror those seen in juvenile NCL patients. There is a decrease in signal intensity ratio in grey matter regions including cortex, hippocampus and cerebellum, tissues where neuronal storage accumulation and cell loss have been seen in the mouse model. The alterations seen in Cln3 mutant mice support the validity of further imaging studies and suggest that this method will have application in assessment of therapeutic approaches in the study of mutant mouse models of NCL including the Cln3 mouse.

Animals↗
PubMed2001

Identification of a transactivation motif in the CLN3 protein.

A transactivation motif has been identified in the neurodegenerative disease protein, CLN3. The C-terminal domain (residues 394-438) of CLN3 can function as a transcriptional activator when fused to the DNA binding domain, LexA. A series of deletion and substitution constructs have been generated to identify the essential region for transactivation. A similar motif is also present in the POU domain transcription factor, nubbin. However, this domain alone does not activate transcription, allowing further localisation of the critical residues in CLN3 required for activity.

Amino Acid Motifs↗
PubMed2000

Neural tube defects: prevention by folic acid and other vitamins.

Folic acid has been demonstrated in clinical trials to reduce significantly the recurrence (and probably occurrence) of neural tube defects (NTD). In the U.K., there has been no decline in prevalence of NTD since the publication of the findings with folic acid. This article examines a series of questions relating to the action of folic acid, with emphasis on the use of mouse models as a source of experimental information which cannot easily be obtained by direct study of humans. Several mouse genetic NTD models exhibit sensitivity to prevention by folic acid, whereas other mice which develop morphologically similar NTD are resistant. Folic acid normalises neurulation in the sensitive mouse strains, providing evidence for a direct effect on the developing embryo, not on the pregnant female: Mouse studies do not support the proposed action of folic acid in encouraging the in utero demise of affected fetuses (i.e. terathanasia). Polymorphic variants of several folate-related enzymes have been shown to influence risk of NTD in humans and an inherited abnormality of folate metabolism has been demonstrated in one mouse NTD model. However, the biochemical basis of the action of folic acid in preventing NTD remains to be determined in detail. NTD in one folate-resistant mouse strain can be prevented by myo-inositol, both in utero and in vitro, raising the possibility of a therapeutic role also in humans. Gene-gene interactions seem likely to underlie the majority of NTD, suggesting that poly-therapy involving folic acid and other agents, such as myo-inositol, may prove more effective in preventing NTD than folic acid treatment alone.

Animals↗
PubMed1999

Developmental expression of palmitoyl protein thioesterase in normal mice.

Deficiency in palmitoyl protein thioesterase (PPT) results in the rapid death of neocortical neurons in human. Very little is known about the developmental and cell-specific expression of this lysosomal enzyme. Here we show that PPT is expressed as a major 2.65 kb and a minor 1.85 kb transcript in the mouse brain. Transcript levels gradually increase between postnatal days 10 and 30. In situ hybridization analysis revealed that PPT transcripts are found widely but not homogeneously in the brain. The most intense signal was detected in the cerebral cortex (layers II, IV-V), hippocampal CA1-CA3 pyramidal cells, dentate gyrus granule cells and the hypothalamus. Immunostaining of PPT was localized in the cell soma, axons and dendrites, especially in the pyramidal and granular cells of the hippocampus, correlating well, both spatially and temporally, with the immunoreactivity of a presynaptic vesicle membrane protein, synaptophysin. In whole embryos, at embryonic day 8, the PPT mRNA expression was most apparent throughout the neuroepithelium, and from day 9 onwards it was seen in all tissues. The expression pattern of PPT suggests its general significance for the brain cells and reflects the response to maturation and growth of the neural networks. Strong PPT immunoreactivity in the axons and dentrites would imply that PPT may not be exclusively a lysosomal enzyme. A notable correlation with synaptophysin would suggest that PPT may have a role in the function of the synaptic machinery.

Aging↗
PubMed1999

A murine model for juvenile NCL: gene targeting of mouse Cln3.

JNCL is a neurodegenerative disease of childhood caused by mutations in the CLN3 gene. A mouse model for JNCL was created by disrupting exons 1-6 of Cln3, resulting in a null allele. Cln3 null mice appear clinically normal at 5 months of age; however, like JNCL patients, they exhibit intracellular accumulation of autofluorescent material. A second approach will generate mice in which exons 7 and 8 of Cln3 are deleted, mimicking the common mutation in JNCL patients.

Animals↗
PubMed1999

The molecular basis of GROD-storing neuronal ceroid lipofuscinoses in Scotland.

Two distinct clinical subtypes of neuronal ceroid lipofuscinosis caused by mutations in the PPT gene, INCL and vJNCL/GROD, occur at a high frequency in the central region of Scotland. In this paper we summarize the clinical details and the molecular basis underlying the disease in the Scottish patients. Comparison of the combination of mutations in the different clinical types reveals a clear genotype-phenotype correlation.

Adolescent↗
PubMed1999

Targeted disruption of the Cln3 gene provides a mouse model for Batten disease. The Batten Mouse Model Consortium [corrected].

Batten disease, a degenerative neurological disorder with juvenile onset, is the most common form of the neuronal ceroid lipofuscinoses. Mutations in the CLN3 gene cause Batten disease. To facilitate studies of Batten disease pathogenesis and treatment, a murine model was created by targeted disruption of the Cln3 gene. Mice homozygous for the disrupted Cln3 allele had a neuronal storage disorder resembling that seen in Batten disease patients: there was widespread and progressive intracellular accumulation of autofluorescent material that by EM displayed a multilamellar rectilinear/fingerprint appearance. Inclusions contained subunit c of mitochondrial ATP synthase. Mutant animals also showed neuropathological abnormalities with loss of certain cortical interneurons and hypertrophy of many interneuron populations in the hippocampus. Finally, as is true in Batten disease patients, there was increased activity in the brain of the lysosomal protease Cln2/TPP-1. Our findings are evidence that the Cln3-deficient mouse provides a valuable model for studying Batten disease.

Animals↗
PubMed1998

Abnormalities of floor plate, notochord and somite differentiation in the loop-tail (Lp) mouse: a model of severe neural tube defects.

Mouse embryos homozygous for the loop-tail (Lp) mutation fail to initiate neural tube closure at E8.5, leading to a severe malformation in which the neural tube remains open from midbrain to tail. During initiation of closure, the normal mouse neural plate bends sharply in the midline, at the site of the future floor plate. In contrast, Lp/Lp embryos exhibit a broad region of flat neural plate in the midline, displacing the sites of neuroepithelial bending to more lateral positions. Sonic hedgehog (Shh) and Netrin1 are expressed in abnormally broad domains in the ventral midline of the E9.5 Lp/Lp neural tube, suggesting over-abundant differentiation of the floor plate. The notochord is also abnormally broad in Lp/Lp embryos with enlarged domains of Shh and Brachyury expression. The paraxial mesoderm shows evidence of ventralisation, with increased expression of the sclerotomal marker Pax1, and diminished expression of the dermomyotomal marker Pax3. While the expression domain of Pax3 does not differ markedly from wild-type, there is a dorsal shift in the domain of Pax6 expression in the neural tube at caudal levels of Lp/Lp embryos. We suggest that the Lp mutation causes excessive differentiation of floor-plate and notochord, with over-production of Shh from these midline structures causing ventralisation of the paraxial mesoderm and, to a lesser extent, the neural tube. Comparison with other mouse mutants suggests that the enlarged floor plate may be responsible for the failure of neural tube closure in Lp/Lp embryos.

Animals↗
PubMed1997

Inositol prevents folate-resistant neural tube defects in the mouse.

Clinical trials demonstrate that up to 70% of neural tube defects (NTDs) can be prevented by folic acid supplementation in early pregnancy, whereas the remaining NTDs are resistant to folate. Here, we show that a second vitamin, myo-inositol, is capable of significantly reducing the incidence of spinal NTDs in curly tail mice, a genetic model of folate-resistant NTDs. Inositol increases flux through the inositol/lipid cycle, stimulating protein kinase C activity and upregulating expression of retinoic acid receptor beta, specifically in the caudal portion of the embryonic hindgut. This reduces the delay in closure of the posterior neuropore, the embryonic defect that is known to lead directly to spina bifida in curly tail embryos. Our findings reveal a molecular pathway of NTD prevention and suggest the possible efficacy of combined treatment with folate and inositol in overcoming the majority of human NTDs.

Animals↗
PubMed1997

Mechanisms of normal and abnormal neurulation: evidence from embryo culture studies.

The method of whole embryo culture has been used extensively in analyzing the mechanisms underlying formation of the mammalian neural tube. These studies have provided insight into the cell lineage of the various tissues that comprise the neurulation stage embryo, the role of microfilaments, extracellular matrix and cell proliferation in the morphogenetic events of neural tube closure and the action of specific genes and gene products in establishment of the nervous system. This information is of considerable importance not only as a means of elucidating the processes of normal embryogenesis but also to shed light on the pathogenesis of important human birth defects.

Animals↗
PubMed1982

Problems in the measurement of putative serum immune complexes by the method of Beaumont.

A methodological investigation of the procedure used by Beaumont et al for measuring a "serum immune complex" precipitated by 25% ammonium sulfate and alleged to contain an ethynyl-estradiol binding immunoglobulin has found major problems with reproducibility and with the correlation of total protein as measured by the Lowry method and the IgG content as determined by a specific nephelometric procedure.

Antigen-Antibody Complex↗